In the state of Alabama, I-65 passes through or near four of the state's major metropolitan areas: Mobile, Montgomery, Birmingham, and Huntsville. I-65 begins its path northward in Mobile at its junction with I-10. From I-10, I-65 runs west of downtown Mobile and through the northern suburbs of the city before turning northeasterly towards Montgomery. In Montgomery, I-65 connects with the southern terminus of I-85. In Birmingham, I-65 has an interchange with I-20/I-59. Sometime in the near future, I-22 will branch off I-65 north of downtown towards Memphis. From Birmingham, I-65 continues north, crossing the Tennessee River near Decatur. A few miles north of the river, it interchanges with I-565, which provides access to Huntsville. It then continues northwards out of the Tennessee Valley to the state of Tennessee, towards Nashville.

At one time, the stretch of I-65 from Louisville to Elizabethtown was a toll road bearing the Kentucky Turnpike name. The bonds that financed the road have been paid off, and tolls are no longer collected. All signs of the former turnpike have been removed.

On November 15, 2006, the stretch of I-65 from Bowling Green to Louisville was renamed the Abraham Lincoln Memorial Highway.

On July 15, 2007, Kentucky highway officials raised its speed limits on Interstate and State Parkway highways to 70 miles per hour (110 km/h). Until that date, Kentucky was the only state along I-65's path that had a speed limit of 65 mph (105 km/h).

Through 2016, the Ohio River Bridges Project is constructing a new six-lane suspension bridge (eventually all-northbound) at Louisville and rebuilding the I-65/I-64/I-71 convergence interchange just south of I-65's existing (1963) John F. Kennedy Memorial Bridge, to become six lanes all-southbound after completion of the new bridge and the Kennedy Bridge re-hab. Another six-lane suspension bridge is also under construction 12 miles (19 km) upstream on the Ohio, to complete the I-265 loop around Louisville.

I-65 enters Indiana at Jeffersonville and Clarksville. Miles 0–9 were rebuilt, widened and realigned from north of Sellersburg to the Ohio River during 2008–10, giving great traffic relief to the fast-growing Indiana suburbs of Louisville. Over 300,000 of the 1.5 million persons in Louisville's CMSA live in its Indiana counties.

The section of I-65 in downtown Indianapolis overlaps I-70. The junctions are often referred to as the "North Split" and the "South Split", forming a section of Interstate locally known as the "Inner Loop" or "The Spaghetti Bowl" due to the visual complexity of the overlapping freeways.

In mid-March 2007, a 6-mile (9.7 km) section of I-70 from the North Split to I-465 east of downtown was restricted to automobiles only for the "Super 70" project, a massive rebuild and expansion of that freeway.[4] Trucks over 13 short tons (12 t) were forced to divert through I-65 if coming from the north and use the circular I-465 to the south to reconnect to I-70 eastbound. Westbound traffic from I-70 was required to loop north or south along I-465 to get to I-65 or I-70. The Super 70 project was completed in November 2007.

In the middle of 2003, the portion of I-65 that runs concurrently with I-70 was closed to all traffic due to the "HyperFix" project. During that time, a new concrete surface was installed and the overpasses were upgraded.

Upon crossing into Lake County, Indiana, over the Kankakee River, the highway is known as the Casimir Pulaski Memorial Highway. It is known as this from that point to its northern terminus.

The northern terminus of Interstate 65 was only 1⁄8 mile (0.20 km) north of I-90 (Indiana Toll Road), prior to 2004. Until then, traffic going from I-90 to I-65 had to make a physical left turn onto I-65 via a traffic signal. Traffic from I-65 to I-90 bypassed the traffic signal via an isolated right-turn lane. In 2004 it was fully grade-separated, so it is now considered to be a single interchange between I-65, I-90, US 12, and US 20, thereby eliminating a connection gap in the Interstate Highway system.